Congenital heart defects (CHDs) affect almost 1% of all live human births and frequently require intervention in order to prevent death. Although the deleterious consequences of such cardiac malformations are usually evident after birth, the causes of these congenital defects frequently involve disregulation of events within the transcriptional programs that control cardiac specification, patterning, differentiation and morphogenesis. Handl and Hand2 are evolutionary conserved basic Helix-Loop-Helix (bHLH) transcription factors that exhibit partially overlapping spatiotemporal expression patterns during cardiac development. Handl and Hand2 are initially co-expressed, but following looping; Handl is predominantly restricted to predominantly the left ventricle, whereas Hand2 to the right ventricle. However, both genes remain co-expressed in the aortic sac, outflow tract (OFT) and the interventricular septum. To test whether Handl and Hand2 have distinct biological functions, we generated Hand1-to-Hand2 knockin chimeric pups. High percentage chimeras die at birth and exhibit hypoplastic left and right ventricles, double-outlet right ventricle (DORV), and both muscular and membranous interventricular septal defects (VSDs). The observed phenotypes occur specifically where endogenous Handl is expressed, suggesting that primary defects in Hand-mediated chamber patterning during early heart development is the root cause of these neonatal CHDs. These data support our hypothesis that Handl and Hand2 convey unique transcriptional regulation during cardiogenesis. In addition to differential expression, Hand proteins can also dimerize with a number of potential bHLH partners (E-proteins, Twist-family, and Hey2) providing yet another regulatory mechanism. Our goals are to identify the distinct functional domains in Handl and Hand2 proteins that convey the observed unique functions and to gain a better understanding of the molecular mechanism/s that underlie the CHDs observed in the Hand1-to-Hand2 knockin pups. Specific Aim 1:Will determine if the cardiovascular anomalies in the Hand1-to-Hand2 mutants are the result of alterations in cell proliferation, survival, or specification of the left and right ventricular cardiomyocytes and/or colonizing cardiac neural crest. Specific Aim 2 will determine what motifs distinguish Handl and Hand2 function and how Handl and 2 functionally interact with Hey2.